Collagen-chitosan-hydroxyapatite composite scaffolds for bone repair in ovariectomized rats

Lesions with bone loss may require autologous grafts, which are considered the gold standard; however, natural or synthetic biomaterials are alternatives that can be used in clinical situations that require support for bone neoformation. Collagen and hydroxyapatite have been used for bone repair based on the concept of biomimetics, which can be combined with chitosan, forming a scaffold for cell adhesion and growth. However, osteoporosis caused by gonadal hormone deficiency can thus compromise the expected results of the osseointegration of scaffolds. The aim of this study was to investigate the osteoregenerative capacity of collagen (Co)/chitosan (Ch)/hydroxyapatite (Ha) scaffolds in rats with hormone deficiency caused by experimental bilateral ovariectomy. Forty-two rats were divided into non-ovariectomized (NO) and ovariectomized (O) groups, divided into three subgroups: control (empty defect) and two subgroups receiving collagen/chitosan/hydroxyapatite scaffolds prepared using different methods of hydroxyapatite incorporation, in situ (CoChHa1) and ex situ (CoChHa2). The defect areas were submitted to macroscopic, radiological, and histomorphometric analysis. No inflammatory processes were found in the tibial defect area that would indicate immune rejection of the scaffolds, thus confirming the biocompatibility of the biomaterials. Bone formation starting from the margins of the bone defect were observed in all rats, with a greater volume in the NO groups, particularly the group receiving CoChHa2. Less bone formation was found in the O subgroups when compared to the NO. In conclusion, collagen/chitosan/hydroxyapatite scaffolds stimulate bone growth in vivo but abnormal conditions of bone fragility caused by gonadal hormone deficiency may have delayed the bone repair process.


Results and discussion
Analysis of the scaffolds. Figure 1 shows the scaffolds and their respective morphologies obtained with the different methods of hydroxyapatite incorporation. The macro images showed no visual differences between the two scaffolds, both presented as three-dimensional structures of white color, similar size, and porous and homogeneous aspect. The porous structures observed in SEM images showed that hydroxyapatite (arrows) was present and distributed throughout the polymeric matrix in both cases, first indication that both adopted hydroxyapatite incorporation methods were successful. Bertolo et al. (2019) found that, although not significant, CoChHa1 scaffolds had pores with smaller diameters (17.7 ± 6.8 µm) than CoChHa2 scaffolds (21.1 ± 8.5 µm) 6 . Moreover, the distribution of these pores was more homogeneous for CoChHa2 (around 33.33% of the pores with 20-30 µm of diameter), indicating that the method of phosphate incorporation into the scaffolds had a great influence on their morphological properties, which may affect the regenerative behavior of the scaffolds in vivo 6 . The pores of the scaffolds used in this study were significantly smaller than those found by Rahman et al. (2019), who prepared scaffolds of rabbit collagen, shrimp chitosan and bovine hydroxyapatite for restoration of defected maxillofacial mandible bone and found pores ranging from 101.69 ± 17 µm to 273.43 ± 49 µm 39 . Macroscopic and radiologic analysis of the bone defect. Macroscopic analysis of the defect area in all rats showed good healing of the soft tissues, as demonstrated by the absence of necrosis, cysts, fibromatosis, abscesses and any evidence of a subcutaneous or deeper tissue inflammatory process. In the bone area, there were also no signs of osteomyelitis, secondary fractures or pseudarthrosis that would suggest any infectious complications (Fig. 2). These good outcomes of the animals may be explained by the fact that the experimental protocol followed the ARRIVE guidelines and principles of the NC3Rs initiative. The animals were monitored for the expression of pain by observing whether the animal was apathetic, depressed, aggressive, or overexcited, such traits being variable in their usual behavior. Possible changes in gait, posture or facial expression were also observed, and appearance, water intake, feeding and clinical symptoms were investigated. There were no complications that needed to be reported and no diseases or signs recommending the removal of an animal (clinical outcome) were observed 40 . In addition, our research group has experience in the method used, as demonstrated by previously published studies 11,[41][42][43] .
The radiographic images showed a round radiolucency of the defect area in the proximal tibial metaphysis and radiopacity of the cortical margins of the tibia, demonstrating the absence of deformities or any other type of change in the bone structure (Fig. 2). These results are compatible with studies on the tibial metaphysis, an experimental model widely used for the investigation of bone repair and biomaterial filling, especially in non-critical defects 44,45 . Thus, the two types of scaffolds used in this study showed biocompatibility in critical bone defects, confirming the biocompatibility of the materials used, both polymers and the inorganic phase incorporated on them 19 . Zugravu et al. (2012) also reported this advantage for collagen/chitosan/hydroxyapatite scaffolds in in vitro studies 46 . Furthermore, the findings demonstrated that gonadal hormone deficiency did not exacerbate the local inflammatory response. www.nature.com/scientificreports/ Morphological analysis of the bone defect area. The formation of new bone in the defect experimentally induced in the proximal tibial metaphysis of rats was observed in all groups analyzed; however, its morphology differed between the control groups and the experimental groups treated with the biomaterial and submitted to ovariectomy. The new bone projected from the margins of the defect but exhibited peculiar histological characteristics that differed between the groups studied and from the original bone of each rat. New formed bone contained lacunae filled with osteocytes that were arranged in various directions. The medullary canal was preserved and filled with hematopoietic tissue and bone trabeculae. Resorption of the biomaterials differed between the grafted groups ( Fig. 3). There is still a preference for the use of autologous grafts for bone grafting; however, due to the need for two surgical beds (donor and recipient area), morbidity and limited availability, advantages arise in the use of synthetic biomaterials for bone tissue regeneration 47 . Three-dimensional scaffolds are particularly interesting in tissue engineering since they can act as structures to accommodate cells and support tissue growth, providing support for cell adhesion, proliferation, and migration 48 . The creation of a bone defect triggers a sequence of events in the local microenvironment, including the migration of inflammatory and proliferative cells of bone tissue, compatible with the remodeling process. These events allow the bone to respond and to adapt to functional changes, as observed in the present study 49 . The resorption rate of biomaterials differs depending on the material used. For example, particulate dentin grafts are characterized by a high resorption rate after 24 months as well as by bone substitution without inflammation. Since dentin particles have open tubes, capillaries can access their interior, resulting in rapid resorption 50 .
There is currently increasing interest in composites consisting of natural polymers (like collagen and chitosan) and hydroxyapatite, which form biocompatible scaffolds with interconnected pores that have a satisfactory osteogenic potential 39,51,52 . Collagen type I is the main component of the extracellular matrix and bone tissue in humans, while hydroxyapatite is the second most abundant component in bone 39 , which can be prepared synthetically with specific nano-sized pores for adequate deposition in the 3D microarchitecture of the scaffolds, called nano-calcium phosphate 6 .
Collagen matrices containing nano-calcium phosphate have shown osteogenic potential in critical bone defects 53 . Recently, synthetic calcium phosphate has been reported to stimulate biomineralization in collagenbased bone substitutes 54 . There is also evidence that chitosan plays an important role in the strengthening of the www.nature.com/scientificreports/ microarchitecture of these polymer matrices, whose biodegradability can be adapted according to the proportion of chitosan or hydroxyapatite 52,55 . Specifically, in non-ovariectomized rats (G1, G2 and G3), the formation of irregular and immature bone was observed in the control group (G1, NO-C), including joining of the margins of the bone defect that contained cavities and spaces but without the interposition of connective tissue. In G2 (NO-CoChHa1), remnants of the biomaterial surrounded almost entirely by newly formed bone were found; however, bone neoformation was not sufficient to fill the entire defect due to the presence of connective tissue in the defect area. In G3 (NO-CoChHa2), the defect closed due to the volume of new bone formed, in a more compact way and promoting the union of the lesion margins, without the presence of connective tissue. Also in G3, there were remnants of the biomaterial inside the medullary canal, which, in turn, was filled with hematopoietic tissue, and several bone trabeculae. This feature was only observed in this group (Fig. 3). These microscopic findings agree with studies on bone repair that report the gradual centripetal substitution of the implanted biomaterial with newly formed bone, demonstrating the biocompatibility of the scaffolds used in this experiment [56][57][58] .
Histological analysis indicated a superior osteogenic potential of CoChHa2, a scaffold in which calcium phosphate was incorporated ex situ into the mixture of collagen gel and chitosan powder. The SEM images of this scaffold had already revealed a porous morphology suitable for growth and cell proliferation. The more homogeneous pore distribution might have been a factor to improve the osteogenic potential of CoChHa2, which was also about 7% less porous and absorbed around 500% less phosphate buffer saline (PBS) than CoChHa1 scaffolds. Moreover, X-rays diffraction patterns of hydroxyapatite (2θ = 32°) were present in both diffractograms in the study of Bertolo et al. (2019) 6 ; however, hydroxyapatite influence on bone formation might have been greater www.nature.com/scientificreports/ in CoChHa2, that presented more intense and better-defined peaks (i.e., greater crystallinity, less influenced by collagen and chitosan presence). According to the histological results, CoChHa2 stimulated greater bone neoformation in the tibial defect area of the animals. Regarding CoChHa1, bone neoformation also occurred around the defect area, but to a lesser extent. In conclusion, the two scaffolds studied here can be indicated for bone repair, since they both present interconnected pores and three-dimensional structures with proven presence of hydroxyapatite; however, there are differences in the time and rate of bone formation, factors directly related to the morphological and physicochemical properties (porosity, absorption, degradation) of the materials, as well as with the availability of the calcium phosphate phase. Scaffolds with more homogeneous porous and with hydroxyapatite more available in the polymeric matrix tend to accelerate the osteogenic process, which is a good feature when working with short recovery periods.
In ovariectomized rats, the formation of thinner bone along the defect was observed in G4 (O-C); however, the space was not completely closed given the presence of connective tissue. In G5 (O-CoChHa1), the young bone was compact at the margins of the defect but more trabecular and bordering the lower surface of the biomaterial which, in turn, was not completely reabsorbed. In G6 (O-CoChHa2), the biomaterial remained intact, with little bone formation around it, and a predominance of connective tissue was thus observed (Fig. 3). Analysis of the scaffolds in ovariectomized rats treated with CoChHa1 (G5) and CoChHa2 (G6) showed less bone formation in the two groups when compared to the respective non-ovariectomized groups; nevertheless, both scaffolds exerted a satisfactory effect as demonstrated by the onset of bone repair, although the process was slower. Evaluating polymer scaffolds in the femur of ovariectomized rats, Cunha et al. (2008) concluded that bone formation is dependent on the properties of the biomaterials as well as on the quality of the host bone tissue 59 .
Preclinical studies using an experimental model similar to that employed in this study have attempted to improve the formation of new bone in rats submitted to ovariectomy (or other models of osteoporosis induction) using tissue engineering techniques. Zhang et al. (2022) developed a new class of copper-alloyed titanium (TiCu) alloys with excellent mechanical properties and biofunctionalization 60 . The authors induced osteoporosis in rats to study the effect of osseointegration and the underlying mechanism of TiCu. The alloy increased fixation stability, accelerated osseointegration, and thus reduced the risk of aseptic loosening during long-term implantation in the osteoporosis environment. The study of  was the first to report the role and mechanism of a copper-alloyed metal in promoting osseointegration in the osteoporosis environment 60 .
Current technologies permit to connect different active functional groups by modifying their configuration or surface. These changes can significantly broaden the range of applications and efficacy of chitosan polymers 61 . Chitosan, calcium phosphate and collagen and their combination in composite materials meet the required properties (biocompatible, bioactive, biodegradable, and multifunctional) and can promote biostimulation for tissue regeneration 52 . In some situations of alteration by pathologies, the repair process and healing rate may be compromised even when these biomaterial are used 62,63 .
Staining with Picrosirius red under polarized light showed collagen birefringence in the extracellular matrix of tissue present in the defect area of all groups (Fig. 4). Picrosirius red is a dye that selectively stains connective tissue, enabling the qualitative analysis of collagen fibers. When observed under polarized light, this stain permits to differentiate especially type I and type III fibers based on the difference in interference colors, intensity and birefringence of the stained tissues 64 . New bone formation was characterized by red-orange birefringence, These results demonstrate that the biomaterials were unable to contribute to bone volume gain when a hormonal component resulting from experimental ovariectomy is involved. On the other hand, the bone volume increase was significant in non-ovariectomized animals grafted with the CoChHa2 scaffold (collagen/chitosan/ hydroxyapatite, method 2). The success of biomaterials for fracture fixation in osteoporotic patients, or simply for bone augmentation, is compromised by poor bone quality and decreased osteoblastic activity. Further research, driven by clinical demand, is therefore needed to address this issue. The growing elderly population and the different associated pathologies require studies that involve close cooperation between basic research, applied research, clinical research, and regulatory bodies 66 .

Conclusions
This in vivo study aimed to evaluate the osteoregenerative capacity of collagen/chitosan/hydroxyapatite scaffolds, obtained by two different methods of hydroxyapatite preparation, in defects created in healthy tibial bone of rats and in fragile bone due to gonadal hormone deficiency (ovariectomy). Macroscopic, radiologic, histomorphological, and morphometric assessment showed that the new bone projected from the margins of the defect in a centripetal manner in all animals. In ovariectomized rats, the new bone forming along the defect was thinner and did not completely close the surgical cavity, as indicated by the presence of connective tissue 5 weeks after surgery.
In non-ovariectomized animals, there was a significant increase in new bone in G3 that received the CoChHa2 scaffold. Thus, this type of scaffold was the best option for in vivo bone growth; however, CoChHa1 can also be used for bone repair, with differences in the time and rate of bone formation. The abnormal conditions of bone fragility caused by gonadal deficiency can delay the process of bone repair, even when scaffolds are used in the search for cell growth and proliferation.

Materials and methods
Collagen was extracted from bovine tendon purchased at Casa de Carnes Santa Paula, Sao Carlos, SP. For chitosan preparation, squid pens (Doryteuthis spp.) were used as a source of β-chitin (obtained at Miami Comércio e Exportação de Pescados Ltda, Cananéia, SP, Brazil). All solvents and reagents used for the preparation of the scaffolds were of analytical grade. Preparation and characterization of collagen/chitosan/hydroxyapatite scaffolds. Two different methods were adopted to prepare the collagen/chitosan/hydroxyapatite scaffolds, according to the methodology described by Bertolo et al. (2019) 6 . The main difference between the methods concerns the in situ (i.e., in the polymeric matrix) or ex situ preparation of calcium phosphate, as described in detail below. In both methods, the collagen: chitosan ratio was kept at 1:1 and the amount of calcium phosphate was 35% of the dry polymer mass in the scaffolds.
CoChHa1-collagen/chitosan/hydroxyapatite, method 1. For calcium phosphate formation, CaCl 2 and (NH 4 ) 2 HPO 4 salts (0.2 mol L −1 and 0.12 mol L −1 , respectively) were added to the 1% (w/w) chitosan gel. After 24 h, the pH of the gel was raised to 9.0 with 1.0 mol L −1 NH 4 OH. After 48 h of stirring at room temperature, excess salts were removed with water and the chitosan solution containing the calcium phosphate synthesized in situ was solubilized in HAc, pH 3.5. Finally, the solution was added to the 1% (w/w) collagen gel and homogeneous mixtures were obtained after 48 h under stirring. After removing the air, the mixtures were placed in Teflon ® molds, frozen, and lyophilized to obtain the scaffolds. The scaffolds were washed in phosphate-buffered saline (PBS), pH 7.4, and in deionized water, frozen, and lyophilized.
CoChHa2-collagen/chitosan/hydroxyapatite, method 2. In this method, calcium phosphate was synthesized ex situ with CaCl 2 .2H 2 O and (NH 4 ) 2 HPO 4 salts (0.05 mol L −1 and 0.03 mol L −1 , respectively) in a 0.15% pectin solution. After calcination to eliminate the pectin matrix, the synthesized calcium phosphate (grain size of 21.4 nm) was added to the 1% (w/w) collagen gel and the mixture was kept under stirring for 60 min. Next, chitosan powder was added to the mixture, which was kept under stirring for 24 h. After removing the air, the mixtures were placed in Teflon ® molds, frozen, and lyophilized to obtain the scaffolds. The scaffolds were washed in PBS, pH 7.4, and in deionized water, frozen, and lyophilized.

Scanning electron microscopy (SEM). CoChHa1 and CoChHa2 morphology was observed with a Zeiss
LEO 440 (Cambridge, England) equipment, with an Oxford detector (model 7060) and an electron beam of   10 . After the confirmation of anesthesia, the animals were placed in ventral decubitus for shaving and asepsis of the lumbar region with 2% chlorhexidine digluconate. A bilateral skin incision was made in the lumbar spine, elevating the skin and muscles in order to expose the retroperitoneum for removal of the ovaries. After removal, the soft tissues and skin were sutured (Ethicon ® , Johnson & Johnson, São José dos Campos, SP, Brazil). In ovariectomized rats, surgery for creation of the proximal metaphyseal defect in the left tibia was performed 5 months after removal of the ovaries. This interval is sufficient to cause bone mineral loss due to estrogen deficiency 59,68 (Fig. 6).

Surgical technique for creation of a proximal metaphyseal defect in the left tibia. Rats of all
groups (G1 to G6) were anesthetized following the same protocol as used for the ovariectomy procedure but adding a subcutaneous dorsal application of Tramadol (2% Cronidor, Brazil). The animals were placed in dorsal decubitus for shaving and asepsis of the left hind leg with 2% chlorhexidine digluconate. After this step, a longitudinal skin incision was made to expose and separate the proximal muscles of the left leg. Once the proximal metaphysis of the left tibia was exposed, a bone defect measuring 2.5 mm in diameter was created with a trephine drill coupled to the pen of a mini-motor (Beltec LB-100, Brazil) until the medullary canal was reached. During this procedure, the area was continuously irrigated with saline to prevent local heating. The contents of the medullary cavity were curetted and the surgical site was washed with saline in order to remove bone remnants resulting from the technical method, thus avoiding any type of osteogenic induction that could compromise the www.nature.com/scientificreports/ histological analysis of bone repair. Next, the biomaterials studied were implanted in G2, G3, G5, and G6, while the bone defect remained empty in the control groups (G1 and G4). After the surgical, the soft tissues including periosteum, muscles, fasciae, and skin were sutured (Fig. 7). In both surgical procedures (ovariectomy and tibial defect creation), rifamycin spray (Rifotrat ® , Brazil) was applied to the surgical site as antibiotic. Each animal then received an intramuscular dose of 0.1 mg/100 g body weight of antibiotic (Pentabiotic Veterinário Pequeno Porte, Fort Dodge ® , Brazil) for one week after surgery. In addition, the rats were constantly monitored and received paracetamol diluted in water in the "feeder" during the postoperative period 69 . Macroscopic, radiologic and histomorphometric analysis of the defect area. Five weeks after surgery, the animals were sacrificed by an overdose of the intramuscular anesthetic, followed by pneumothorax. The left tibia was removed and photodocumented for analysis of the clinical conditions of the defect area. Next, the samples were radiographed to assess the integrity and radiodensity of the defect area.
The left tibiae were kept in formaldehyde solution and were then decalcified to be reduced in the defect areas. The samples were submitted to routine histological methods. Semi-serial 5-µm sections were stained with Masson's trichrome for the characterization of bone neoformation and with Picrosirius red (saturated aqueous solution of picric acid plus 0.1 g Sirius red F3B, Bayer ® , Germany) for the identification of fibrillar extracellular matrix components by polarized light microscopy.
For quantification of the volume of newly formed bone in the defect area of the animals, the Motic Images Plus 2.0 ML software was used to delimit the total area of the bone defect and of newly formed bone. These values were used to obtain the percent volume of bone formed in the defect area. These data were entered into the BioEstat 5.3 software and ANOVA followed by the Tukey test was used for comparison between groups, adopting a level of significance of p < 0.05.

Data availability
The data presented in this study are available on request from the corresponding author.